The biotin-streptavidin coupling of ligands to a biosensor is prior art. A ligand is biotinylated and is immobilized on a sensor surface that is loaded with streptavidin. Analytes to be tested are detected by the binding to the ligands. Disadvantageously, this method requires regeneration steps which are time-consuming and disrupt the non-covalent binding of the ligand to the surface.
Dornieden et al. were able to show, by means of a ThT assay, that the antibody fragment scFv-IC16 hinders Aβ fibril formation in samples containing Aβ 1-42. The same publication described binding a synthetic Aβ 1-42 peptide via its C-terminal cysteine to the sensor surface of a CM5 sensor chip from GE Healthcare. Surface plasmon resonance was carried out in order to characterize the binding behavior of the antibody fragment scFv-IC16 to Aβ conformers. The antibody fragment scFv-IC16 was the analyte in these experiments.
No approved medicament exists for treating the cause of Alzheimer's dementia (AD). Deposits of the so-called beta-amyloid peptide (Aβ) in plaques are typically found post mortem in the brains of AD patients. Various forms of A1, for example fibrils, have been blamed for the onset and progression of AD. For the past few years, small Aβ aggregates (Aβ oligomers) in particular have been blamed as the main culprit for the onset and progression of AD. Reduction or complete elimination of Aβ oligomers would thus appear to be the most important criterion for curing or slowing AD. Aβ monomers are constantly being produced in our body and are presumably not toxic per se. There is speculation as to whether Aβ monomers agglomerate randomly depending on their concentration (which ultimately results from the rate at which they are formed and broken down in the body) and thus are increasingly more likely to form Aβ oligomers spontaneously as a person gets older. Once formed, Aβ oligomers could then multiply through a prion-like mechanism and ultimately lead to the disease. Based on these considerations, causal treatment should be aimed at completely destroying toxic Aβ oligomers, and possibly also other oligomer forms, and/or hindering the prion-like multiplication thereof.
One important point is the fact that any active ingredient has to be tested in an animal model and in clinical studies. These are very time-consuming and costly. A rapid, reliable and quantitative in vitro analysis, which permits both a screening of various potential active ingredients as well as the effect of active ingredient optimizations on the specific binding behavior for different Aβ conformers (monomers, oligomers, fibrils), would be of great advantage. In addition, a direct detection of Aβ oligomers and/or other conformers other than monomers at any desired point in time during the measurement is an important requirement for functioning as a control/quality standard.
For quantitative binding analyses of substances, surface plasmon resonance (SPR) technology is primarily used at present since this enables conclusions to be drawn about the underlying association and dissociation rates in addition to binding affinities. For interaction studies, one of the two interaction partners must be immobilized on the sensor surface. This is known as the ligand.
Due to the high susceptibility of Aβ oligomers to changes in structure, use as an analyte (injected molecule) is not advisable. For immobilizing molecules, various covalent and non-covalent strategies currently exist. One particular challenge in the case of Aβ oligomers is the high susceptibility thereof to undergoing structural changes as a result of changes in the surrounding solution conditions, which cannot be avoided in the case of covalent coupling methods. Non-covalent immobilization techniques, so-called capture methods, are the method of choice here. In this case, however, there is a need to find a successful combination of suitable Aβ oligomer preparation methods with non-covalent immobilization methods.
Nevertheless, in various current publications, there is a trend toward covalent immobilization without taking account of resulting possible structural changes in the case of immobilized Aβ oligomers.
Regeneration steps between individual analyte injections are a further source of error in SPR measurements, since these can likewise bring about structural changes.
Detection using antibodies serves as a common detection method for the successful immobilization of Aβ oligomers on sensor surfaces. Due to the bivalent nature of the antibodies, avidity effects disadvantageously occur, which do not allow for a complete dissociation of the molecules in a reasonable length of time, without the addition of additives and again require regeneration steps.
Due to the described high-susceptibility to structural changes as a result of changing solution conditions which are necessary due to conventional immobilization protocols, regeneration steps between individual analyte injections and antibody control injections, measurement artefacts cannot be avoided in interaction studies using oligomeric Aβ forms. This harbors the risk that measurement data obtained are possibly highly subject to errors.
The problem addressed by the invention is that of providing a method for the quantitative characterization of substances with regard to their properties of binding to various amyloid-β (Aβ) conformers. This would permit screening and the optimization of substances to be tested, for example against Alzheimer's dementia (AD). Another problem addressed by the invention is that of providing a device for carrying out the method.